Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity

Abstract

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a novel, kidney-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of renal tubular epithelial cells. To characterize the Ksp-cadherin gene promoter, a lambda bacteriophage clone containing 3.7 kb of the proximal 5' flanking region of the mouse Ksp-cadherin gene was isolated. The transcription initiation site was mapped by RNase protection assays and 5' rapid amplification of cDNA ends, and a 709-bp intron was identified within the 5' untranslated region. The proximal 5' flanking region was "TATA-less" but contained other consensus promoter elements including an initiator (Inr), GC boxes, and a CAAT box. Potential binding sites were identified for transcription factors that are involved in tissue-specific gene expression including activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF-3), basic helix-loop-helix (bHLH) proteins, CCAAT/enhancer-binding protein (C/EBP), and GATA factors. Transfection of luciferase reporter plasmids containing 2.6 kb of the 5' flanking region markedly increased luciferase activity in renal epithelial cells (MDCK and mIMCD-3) but not in mesenchymal cells (NIH 3T3 and MMR1). Deletion analysis identified an 82-bp region from -31 to -113 that was essential for promoter activity in transfected renal epithelial cells. Electrophoretic mobility-shift assays showed that mIMCD-3 cells contain nuclear proteins that bind to this region of the promoter. Mutational analysis showed that sequences within the HNF-3 consensus site and CAAT box were involved in protein binding and promoter activity. We conclude that the proximal 5' flanking region of the mouse Ksp-cadherin gene contains an orientation-dependent promoter that is kidney epithelial cell specific. The region of the promoter from -113 to -31 is required for transcriptional activity and contains binding sites for nuclear proteins that are specifically expressed in renal epithelial cells.

Transcriptome of a mouse kidney cortical collecting duct cell line: Effects of aldosterone and vasopressin

“…Interestingly, putative DNA binding sites have been identified in the promoter regions of several sodium transporters (Na,K-ATPase, Na͞H exchanger) or of the MR (30). The HNF-3-␣ binding site was also identified in the promoter region of a novel, kidney-specific, cell adhesion molecule, cadherin 16 (or Ksp-cadherin), which is highly expressed in the CCD (31). RAMP3 belongs to a recently identified receptor activity modifying protein (RAMP) family of molecular chaperones responsible for the proper glycosylation and transport of the calcitonin receptor-like receptor (CRLR) to the plasma membrane.…”

Cloning of the Human Claudin-2 5′-Flanking Region Revealed a TATA-less Promoter with Conserved Binding Sites in Mouse and Human for Caudal-related Homeodomain Proteins and Hepatocyte Nuclear Factor-1α

“…In contrast HNF family members have been demonstrated to activate tissue type-specific expression of Ksp cadherin (cadherin-16), which lacks TATA boxes (48).…”

Common regulatory elements in the polycystic kidney disease 1 and 2 promoter regions

“…The 300 bp fragment (À127 to þ 157) stimulated luciferase activity in all cell lines, showing high levels of activity in the renal cell lines and lower activity in the colon and liver cell lines (Figure 4). In contrast, a well-characterized 1.3 kb promoter region of the kidney-specific Ksp-Cadherin gene 32 showed promoter activity only in the renal epithelial cell lines, and not in the colon, liver, and HEK293 cells (not shown). To correlate reporter gene expression to endogenous Pkd1 expression, we applied real-time PCR to determine Pkd1 expression in the mousederived cell lines mIMCD3 and mhAT3.…”